PROJECT SUMMARY Myocardial ischemia-reperfusion injury is a leading cause of death in both males and females in the United States. Cardioprotective mechanisms hold promise for lessening this burden, but few experimental discoveries have translated successfully into effective therapeutics. Nitric oxide, produced endogenously or administered exogenously, has been identified as an essential component of many different cardioprotective pathways. Our recent findings suggest that the S-nitrosation (SNO) of cysteine thiols is essential for nitric oxide-dependent protection in female hearts, which exhibit higher baseline SNO levels compared to males and associated with this, greater protection from ischemia-reperfusion injury. SNO levels are regulated by S-nitrosoglutathione reductase (GSNOR), which catabolizes protein SNO, and we have evidence to suggest that GSNOR is a key mediator of protection in the heart. We have also identified two additional targets (TRIM72, mitochondrial proteins) that are critical for the SNO-mediated ischemic stress response in male and female hearts, but specific mechanisms for reducing cell death and potential sex differences are not known. As such, a significant knowledge gap exists regarding a number of fundamental questions related to the physiological and pathophysiological role(s) of SNO signaling in male and female hearts. To address this knowledge gap, we have developed a number of novel methods for the identification and quantitation of specific in situ SNO modification sites in the heart. In the present application, we propose to use these methods in tandem with in vivo and ex vivo models of myocardial ischemia-reperfusion injury and newly developed and emerging methodologies, like in vivo stable isotope labeling by amino acids in mammals (SILAM) to assess protein turnover, quantitative mass spectrometry, and metabolomics analysis. These approaches will be used to enhance our mechanistic understanding of SNO signaling in sex-dependent cardioprotection through the following specific aims: 1) define the role of TRIM72 as a protective target of SNO during ischemia-reperfusion injury, 2) define the protective role of SNO-modified mitochondrial proteins during ischemia-reperfusion injury, and 3) define the role of GSNOR in mitigating nitrosative stress during ischemia-reperfusion injury. If completed successfully, the aims of this proposal will advance our mechanistic understanding of protein SNO signaling in myocardial ischemia- reperfusion injury and cardioprotection. These studies will also provide valuable insight into how SNO-based approaches may be targeted for the therapeutic treatment of ischemic heart disease in both males and females.